Use of adipose tissue-derived stromal cells for chondrocyte differentiation and cartilage repair

ABSTRACT

Methods and compositions for directing adipose-derived stromal cells cultivated in vitro to differentiate into cells of the chondrocyte lineage are disclosed. The invention further provides a variety of chondroinductive agents which can be used singly or in combination with other nutrient components to induce chondrogenesis in adipose-derived stromal cells either in cultivating monolayers or in a biocompatible lattice or matrix in a three-dimensional configuration. Use of the differentiated chondrocytes for the therapeutic treatment of a number of human conditions and diseases including repair of cartilage in vivo is disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/250,742 filed Sep. 30, 2011, now pending; which is a continuationapplication of U.S. application Ser. No. 11/408,381 filed Apr. 21, 2006,now issued as U.S. Pat. No. 8,048,673; which is a continuationapplication of U.S. application Ser. No. 10/980,137 filed Nov. 1, 2004,now issued as U.S. Pat. No. 7,033,587; which is a divisional applicationof U.S. application Ser. No. 10/125,106 filed Apr. 18, 2002, now issuedas U.S. Pat. No. 6,841,150; which is a continuation application of U.S.application Ser. No. 09/573,989 filed May 17, 2000, now issued as U.S.Pat. No. 6,429,013; which claims the benefit under 35 USC §119(e) toU.S. Application Ser. No. 60/149,850 filed Aug. 19, 1999, now expired.The disclosure of each of the prior applications is considered part ofand is incorporated by reference in the disclosure of this application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods and compositions for directingadipose-derived stromal cells cultivated in vitro to differentiate intocells of the chondrocyte lineage and particularly to such directedlineage induction prior to, or at the time of, their implantation into arecipient or host for the therapeutic treatment of pathologic conditionsin humans and other species.

2. Background Information

Mesenchymal stem cells (MSCs) are the formative pluripotent blast orembryonic-like cells found in bone marrow, blood, dermis, and periosteumthat are capable of differentiating into specific types of mesenchymalor connective tissues including adipose, osseous, cartilaginous,elastic, muscular, and fibrous connective tissues. The specificdifferentiation pathway which these cells enter depends upon variousinfluences from mechanical influences and/or endogenous bioactivefactors, such as growth factors, cytokines, and/or localmicroenvironmental conditions established by host tissues.

In prenatal organisms, the differentiation of MSCs into specializedconnective tissue cells is well established; for example embryonicchick, mouse or human limb bud mesenchymal cells differentiate intocartilage, bone and other connective tissues (Caplan A I (1981) In: 39thAnnual Symposium of the Society for Developmental Biology, ed by S.Subtelney and U Abbott, pp 3768. New York, Alan R Liss Inc; Elmer et al.(1981) Teratology, 24:215-223; Hauschka S. D. (1974) DevelopmentalBiology (1974) 37:345-368; Solursh et al. (1981) Developmental Biology,83:9-19; Swalla et al. (1986) Developmental Biology, 116:31-38. Inaddition, a clonal rat fetus calvarial cell line has also been shown todifferentiate into muscle, fat, cartilage, and bone (Goshima et al.(1991) Clin Orthop Rel Res. 269:274-283. The existence of MSCs inpost-natal organisms has not been widely studied with the objective ofshowing the differentiation of post-embryonic cells into severalmesodermal phenotypes. The few studies which have been done involve theformation of bone and cartilage by bone marrow cells following theirencasement in diffusion chambers and in vivo transplantation (Ashton etal. (1980) Clin Orthop Rd Res, 151:294-307; Bruder et al. (1990) BoneMineral, 11:141-151, 1990). Recently, cells from chick periosteum havebeen isolated, expanded in culture, and, under high density conditionsin vitro, shown to differentiate into cartilage and bone (Nakahara etal. (1991) Exp Cell Res, 195:492-503). Rat bone marrow-derivedmesenchymal cells have been shown to have the capacity to differentiateinto osteoblasts and chondrocytes when implanted in vivo (Dennis et al.(1991) Cell Transpl, 1:2332; Goshima et al. (1991) Clin Orthop Rd Res.269:274-283). Work by Johnstone et al. U.S. Pat. No. 5,908,784 has shownthe ability of mesenchymal cells derived from skin to differentiate intocells biochemically and phenotypically similar to chondrocytes.

The adult bone marrow microenvironment is a potential source for thesehypothetical mesodermal stem cells. Cells isolated from adult marrow arereferred to by a variety of names, including stromal cells, stromal stemcells, mesenchymal stem cells (MSCs), mesenchymal fibroblasts,reticular-endothelial cells, and Westen-Bainton cells (Gimble et al.November 1996) Bone 19(5): 421-8). In vitro studies have determined thatthese cells can differentiate along multiple mesodermal or mesenchymallineage pathways. These include, but are not limited to, adipocytes(Gimble, et al. (1992) J. Cell Biochem. 50:73-82, chondrocytes; Caplanet al. (1998) J Bone Joint Surg. Am. 80(12): 1745-57; hematopoieticsupporting cells, Gimble, et al. (1992) J. Cell Biochem. 50:73-82;myocytes, Prockop, et al. (1999) J. Cell Biochem. 72(4):570-85;myocytes, Charbord et al. (1999) Exp. Hematol. 27(12):1782-95; andosteoblasts, Beresford et al. (1993) J. Cell Physiol. 154:317-328). Thebone marrow has been proposed as a source of stromal stem cells for theregeneration of bone, cartilage, muscle, adipose tissue, and othermesenchymal derived organs. The major limitations to the use of thesecells are the difficulty and risk attendant upon bone marrow biopsyprocedures and the low yield of stem cells from this source.

Adipose tissue offers a potential alternative to the bone marrow as asource of multipotential stromal stem cells. Adipose tissue is readilyaccessible and abundant in many individuals. Obesity is a condition ofepidemic proportions in the United States, where over 50% of adultsexceed the recommended BMI based on their height. Adipocytes can beharvested by liposuction on an outpatient basis. This is a relativelynon-invasive procedure with cosmetic effects that are acceptable to thevast majority of patients. It is well documented that adipocytes are areplenishable cell population. Even after surgical removal byliposuction or other procedures, it is common to see a recurrence ofadipocytes in an individual over time. This suggests that adipose tissuecontains stromal stem cells which are capable of self-renewal.

Pathologic evidence suggests that adipose-derived stromal cells arecapable of differentiation along multiple mesenchymal lineages. The mostcommon soft tissue tumor, liposarcomas, develop from adipocyte-likecells. Soft tissue tumors of mixed origin are relatively common. Thesemay include elements of adipose tissue, muscle (smooth or skeletal),cartilage, and/or bone. Just as bone forming cells within the bonemarrow can differentiate into adipocytes or fat cells, theextramedullary adipocytes are capable of forming osteoblasts (HalvorsenWO 99/28444).

Cartilage is a hyperhydrated structure with water comprising 70% to 80%of its weight. The remaining 20% to 30% comprises type II collagen andproteoglycan. The collagen usually accounts for 70% of the dry weight ofcartilage (in “Pathology” (1988) Eds. Rubin & Farber, J. B. LippincottCompany, PA. pp. 1369-1371). Proteoglycans are composed of a centralprotein core from which long chains of polysaccharides extend. Thesepolysaccharides, called glycosaminoglycans, include:chondroitin-4-sulfate, chondroitin-6-sulfate, and keratan sulfate.Cartilage has a characteristic structural organization consisting ofchondrogenic cells dispersed within an endogenously produced andsecreted extracellular matrix. The cavities in the matrix which containthe chondrocytes are called cartilage lacunae. Unlike bone, cartilage isneither innervated nor penetrated by either the vascular or lymphaticsystems (Clemente (1984) in “Gray's Anatomy, 30^(th) Ed.,” Lea &Febiger).

Three types of cartilage are present in mammals and include: hyalinecartilage; fibrocartilage and elastic cartilage (Rubin and Farber,supra). Hyaline cartilage consists of a gristly mass having a firm,elastic consistency, is translucent and is pearly blue in color. Hyalinecartilage is predominantly found on the articulating surfaces ofarticulating joints. It is found also in epiphyseal plates, costalcartilage, tracheal cartilage, bronchial cartilage and nasal cartilage.Fibrocartilage is essentially the same as hyaline cartilage except thatit contains fibrils of type I collagen that add tensile strength to thecartilage. The collagenous fibers are arranged in bundles, with thecartilage cells located between the bundles. Fibrocartilage is foundcommonly in the annulus fibrosis of the invertebral disc, tendinous andligamentous insertions, menisci, the symphysis pubis, and insertions ofjoint capsules. Elastic cartilage also is similar to hyaline cartilageexcept that it contains fibers of elastin. It is more opaque thanhyaline cartilage and is more flexible and pliant. These characteristicsare defined in part by the elastic fibers embedded in the cartilagematrix. Typically, elastic cartilage is present in the pinna of theears, the epiglottis, and the larynx.

The surfaces of articulating bones in mammalian joints are covered witharticular cartilage. The articular cartilage prevents direct contact ofthe opposing bone surfaces and permits the near frictionless movement ofthe articulating bones relative to one another (Clemente, supra). Twotypes of articular cartilage defects are commonly observed in mammalsand include full-thickness and partial-thickness defects. The two typesof defects differ not only in the extent of physical damage but also inthe nature of repair response each type of lesion elicits.

Full-thickness articular cartilage defects include damage to thearticular cartilage, the underlying subchondral bone tissue, and thecalcified layer of cartilage located between the articular cartilage andthe subchondral bone. Full-thickness defects typically arise duringsevere trauma of the joint or during the late stages of degenerativejoint diseases, for example, during osteoarthritis. Since thesubchondral bone tissue is both innervated and vascularized, damage tothis tissue is often painful. The repair reaction induced by damage tothe subchondral bone usually results in the formation of fibro cartilageat the site of the full-thickness defect. Fibrocartilage, however, lacksthe biomechanical properties of articular cartilage and fails to persistin the joint on a long term basis.

Partial-thickness articular cartilage defects are restricted to thecartilage tissue itself. These defects usually include fissures orclefts in the articulating surface of the cartilage. Partial-thicknessdefects are caused by mechanical arrangements of the joint which in turninduce wearing of the cartilage tissue within the joint. In the absenceof innervation and vasculature, partial-thickness defects do not elicitrepair responses and therefore tend not to heal. Although painless,partial-thickness defects often degenerate into full-thickness defects.

In accordance with the present invention it has been observed by theinventors that when human adipose tissue-derived stromal cells areassociated in a three-dimensional format they can be induced to commitand differentiate along the chondrogenic pathway when contacted in vitrowith certain chondroinductive agents or factors. The three dimensionalformat is critical to the in vitro chondrogenesis of the invention andthe cells are preferably condensed together, for example, as a packed orpelleted cell mass or in an alginate matrix. This invention presentsexamples of methods and composition for the isolation, differentiation,and characterization of adult human extramedullary adipose tissuestromal cells along the chondrocyte lineage and outlines their use forthe treatment of a number of human conditions and diseases. This invitro process is believed to recapitulate that which occurs in vivo andcan be used to facilitate repair of cartilage in vivo in mammals.

SUMMARY OF THE INVENTION

The present invention provides methods and composition for consistentand quantitative induction of stromal cells derived from subcutaneous,mammary, gonadal, or omental adipose tissues into fully functionalchondrocytes. The methods comprise incubation of isolated adiposetissue-derived stromal cells, plated at densities of 500 to 20,000cells/cm² in a chemically defined culture medium having or supplementedwith (1) a chondroinductive agent that can activate any cellulartransduction pathway leading to the mature chondrocyte phenotype; (2) anantibiotic; (3) a nutrient supplement such as fetal bovine serum orhorse serum; (4) ascorbate or related vitamin C analogue; and (5) aglucocorticoid or another chemical agent capable of activating thecellular glucocorticoid receptor.

The present invention also provides a method for differentiating adiposetissue derived stromal cells into chondrocytic cells by pelletingstromal cells in medium such as DMEM or alpha-MEM or RPMI 1640 andsupplementing the medium with (1) a chondroinductive agent that canactivate any cellular transduction pathway leading to the maturechondrocyte phenotype; (2) an antibiotic; (3) a nutrient supplement suchas fetal bovine serum or horse serum; (4) ascorbate or related vitamin Canalogue; and (5) a glucocorticoid or another chemical agent capable ofactivating the cellular glucocorticoid receptor.

The present invention also provides a method for differentiating adiposetissue derived stromal cells into chondrocytic cells by suspending thecells in a calcium alginate or other biocompatible lattice or matrixcapable of supporting chondrogenesis in a three dimensionalconfiguration.

The present invention provides methods for determining the ability ofthese culture conditions and agents to direct the differentiation andfunction of the adipose tissue-derived stromal cells, for thetransduction of viral vectors carrying regulatory genes into the stromalcells, for the transfection of plasmid vectors carrying regulatory genesinto the stromal cells, for the tracking and detection of functionalproteins encoded by these genes, and for developing biomechanicalcarriers for the re-introduction of these cells into a living organism.

This invention further provides methods for the introduction of thesechondrocytes into cartilage defect areas for repair.

The methods and composition have use in drug discovery for compounds andproteins with relevance to the differentiated cell-related diseasestates and traumatic injuries including but not limited to: anteriorcrucia ligament tears, full-thickness articular cartilage defects,partial-thickness articular cartilage defects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the immunodetection of collagen type II in human adiposestromal cells from monolayer cultures. Phase contrast microscopy is usedin the upper panel; immunofluorescence is used in the lower panel.

FIG. 2 shows immunodetection of collagen type II in human adiposestromal cells from pellet cultures. Phase contrast microscopy is used inthe upper panel; immunofluorescence is used in the lower panel.

FIG. 3 shows immunodetection of collagen type II in human adiposestromal cells from alginate cultures. Phase contrast microscopy is usedin the upper panel; immunofluorescence is used in the lower panel.

FIG. 4 shows Collagen type VI expression when cells were cultured in analginate matrix at 2 weeks without TGF-beta (control) and with TGF-beta.

FIG. 5 shows a Western blot of results when cells were grown asmonolayers or in an alginate suspension for the expression of differentproteins including: collagen type VI, link, aggrecan, collagen type I,and actin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and a composition for thedifferentiation and culture of adipose tissue-derived stromal cells intochondrocytes. The cells produced by the methods of invention are usefulin providing a source of fully differentiated and functional cells forresearch, transplantation, and development of tissue engineeringproducts for the treatment of human disease and traumatic injury repair.Thus, in one aspect, the invention provides a method for differentiatingadipose tissue-derived stromal cells into chondrocytes comprisingculturing stromal cells in a composition which comprises a mediumcapable of supporting the growth and differentiation of stromal cellsinto functional chondrocytes. This invention further provides methodsfor the introduction of these chondrocytes into cartilage defect areasfor repair.

“Adipose stromal cells” refers to stromal cells that originate fromadipose tissue. By “adipose” is meant any fat tissue. The adipose tissuemay be brown or white adipose tissue, derived from subcutaneous,omental/visceral, mammary, gonadal, or other adipose tissue site.Preferably, the adipose is subcutaneous white adipose tissue. Such cellsmay comprise a primary cell culture or an immortalized cell line. Theadipose tissue may be from any organism having fat tissue. Preferably,the adipose tissue is mammalian, most preferably the adipose tissue ishuman. A convenient source of adipose tissue is from liposuctionsurgery, however, the source of adipose tissue or the method ofisolation of adipose tissue is not critical to the invention. If stromalcells are desired for autologous transplantation into a subject, theadipose tissue will be isolated from that subject.

“Chondrocytes (cartilage cells)” refers to cells that are capable ofexpressing characteristic biochemical markers of chondrocytes, includingbut not limited to collagen type II, chondroitin sulfate, keratinsulfate and characteristic morphologic markers of smooth muscle,including but not limited to the rounded morphology observed in culture,and able to secrete collagen type II, including but not limited to thegeneration of tissue or matrices with hemodynamic properties ofcartilage in vitro.

Any medium capable of supporting stromal cells in tissue culture may beused. Media formulations that will support the growth of fibroblastsinclude, but are not limited to, Dulbecco's Modified Eagle's Medium(DMEM), alpha modified Minimal Essential Medium (αMEM), and Roswell ParkMemorial Institute Media 1640 (RPMI Media 1640) and the like. Typically,0 to 20% Fetal Bovine Serum (FBS) or 1-20% horse serum will be added tothe above media in order to support the growth of stromal cells and/orchondrocytes. However, a defined medium could be used if the necessarygrowth factors, cytokines, and hormones in FBS for stromal cells andchondrocytes are identified and provided at appropriate concentrationsin the growth medium. Media useful in the methods of the invention maycontain one or more compounds of interest, including, but not limited toantibiotics mitogenic or differentiative compounds for stromal cells.The cells will be grown at temperatures between 31° C. to 37° C. in ahumidified incubator. The carbon dioxide content will be maintainedbetween 2% to 10% and the oxygen content between 1% and 22%. Cells willremain in this environment for periods of up to 4 weeks.

Antibiotics which can be supplemented into the medium include, but arenot limited to penicillin and streptomycin. The concentration ofpenicillin in the chemically defined culture medium is about 10 to about200 units per ml. The concentration of streptomycin in the chemicallydefined culture medium is about 10 to about 200 μg/ml.

Glucocorticoids that can be used in the invention include but are notlimited to hydrocortisone and dexamethasone. The concentration ofdexamethasone in the medium is about 1 to about 100 nM. Theconcentration of hydrocortisone in the medium is about 1 to about 100nM.

As used herein the terms “chondroinductive agent” or “chondroinductivefactor” refers to any natural or synthetic, organic or inorganicchemical or biochemical compound or combination or mixture of compounds,or any mechanical or other physical device, container, influence orforce that can be applied to human adipose tissue-derived stromal cellsso as to affect their in vitro chondrogenic induction or the productionof chondrocytes. The chondroinductive agent is preferably selected,individually or in combination, from the group consisting of (i) aglucocorticoid such as dexamethasone; a member of the transforminggrowth factor-β superfamily such as a bone morphogenic protein(preferably BMP-2 or BMP-4), TGF-β1, TGF-β2, TGF-β3, insulin-like growthfactor (IGF), platelet derived growth factor (PDGF), epidermal growthfactor (EGF), acidic fibroblast growth factor (aFBF), basic fibroblastgrowth factor (bFBF), hepatocytic growth factor (HGF), keratocyte growthfactor (KGF), osteogenic proteins (OP-1, OP-2, and OP-3), inhibin A orchondrogenic stimulating activity factor (CSA); (iii) a component of thecollagenous extracellular matrix such as collagen I (particularly in theform of a gel); (iv) a vitamin A analogue such as retinoic acid, and (v)ascorbate or other related vitamin C analogue.

The concentration of transforming growth factor-beta is about 1 to about100 ng/ml. The concentration of retinoic acid is about 0.1 to about 1μg/ml.

Examples of compounds that are stromal cell mitogens include but are notlimited to transforming growth factor β; fibroblast growth factor, bonemorphogenetic protein and stromal cell differentiating factors includebut are not limited to dexamethasone, hydrocortisone, transforminggrowth factor β, fibroblast growth factor, and bone morphogeneticprotein and the like.

Preferably, the adipose tissue derived stromal cells are isolated fromthe adipose tissue of the subject into which the final differentiatedcells are to be introduced. However, the stromal cells may also beisolated from any organism of the same or different species as thesubject. Any organism with adipose tissue can be a potential candidate.Preferably, the organism is mammalian, most preferably the organism ishuman.

The present invention also provides a method for differentiating adiposederived stromal cells into chondrocytic cells by suspending the cells ina calcium alginate or another biocompatible lattice or matrix capable ofsupporting chondrogenesis in a three dimensional configuration. Examplesof lattice materials include (1) calcium alginate, a polysaccharide ofcross-linked L-glucuronic and D-mannuronic acid, at concentrations ofbetween 1% to 4%; (2) fibrin; (3) collagen type II; or (4) agarose gel.The lattices or matrixes containing the cells are transferred to culturedishes containing: (1) a chondroinductive agent that can activate anycellular transduction pathway leading to the mature chondrocytephenotype; (2) an antibiotic; (3) a nutrient supplement such as fetalbovine serum or horse serum; (4) ascorbate or related vitamin Canalogue; and (5) a glucocorticoid or another chemical agent capable ofactivating the cellular glucocorticoid receptor.

The adipose tissue derived stromal cells may be stably or transientlytransfected or transduced with a nucleic acid of interest using aplasmid, viral or alternative vector strategy. Nucleic acids of interestinclude, but are not limited to, those encoding gene products whichenhance the production of extracellular matrix components found incartilage; examples include transforming growth factor β, bonemorphogentic protein, activin and insulin-like growth factor.

The transduction of viral vectors carrying regulatory genes into thestromal cells can be performed with viral vectors (adenovirus,retrovirus, adeno-associated virus, or other vector) purified by cesiumchloride banding or other method at a multiplicity of infection (viralunits:cell) of between 10:1 to 2000:1. Cells will be exposed to thevirus in serum free or serum-containing medium in the absence orpresence of a cationic detergent such as polyethyleneimine orLipofectamine™ for a period of 1 hour to 24 hours (Byk T. et al. (1998)Human Gene Therapy 9:2493-2502; Sommer B. et al. (1999) Calcif. TissueInt. 64:45-49).

The transfection of plasmid vectors carrying regulatory genes into thestromal cells can be introduced into the cells in monolayer cultures byuse of calcium phosphate DNA precipitation or cationic detergent methods(Lipofectamine™, DOTAP) or in three-dimensional cultures byincorporation of the plasmid DNA vectors directly into the biocompatiblepolymer (Bonadio J. et al. (1999) Nat. Med. 5:753-759).

For the tracking and detection of functional proteins encoded by thesegenes, the viral or plasmid DNA vectors will contain a readilydetectable marker gene, such as the green fluorescent protein orbeta-galactosidase enzyme, both of which can be tracked by histochemicalmeans.

For the development of biomechanical carriers for the reintroduction ofthe stromal cells into a living organism, the carriers include but arenot limited to calcium alginate, agarose, types I, II, IV or othercollagen isoform, fibrin, poly-lactic/poly-glycolic acid, hyaluronatederivatives or other materials (Perka C. et al. (2000) J. Biomed. Mater.Res. 49:305-311; Sechriest V. F. et al. (2000) J. Biomed Mater. Res.49:534-541; Chu C. R. et al. (1995) J. Biomed Mater. Res. 29:1147-1154;Hendrickson D. A. et al. (1994) Orthop. Res. 72:485-497).

Another object of the invention is to provide for the identification andstudy of compounds that enhance the differentiation of adipose tissuederived stromal cells into chondrocytes. Compounds which enhancedifferentiation may be of value in the treatment of partial or fullcartilage defects, osteoarthritis, traumatized cartilage, cosmeticsurgery of inborn defects including cleft palate or deviated septum.Methods include but are not limited to the development ofthree-dimensional in vitro cultures maintaining adipose tissue-derivedstromal cells as chondrocytes that can be subsequently exposed to novelcompounds of interest.

Any compound may be tested for its ability to affect the differentiationof adipose tissue derived stromal cells into chondrocytes. Appropriatevehicles compatible with the compound to be tested are known to thoseskilled in the art and may be found in the current edition ofRemington's Pharmaceutical Sciences (1995, Mack Publishing Co., Easton,Pa.) the contents of which are incorporated herein by reference.

The features and advantages of the present invention will be moreclearly understood by reference to the following examples, which are notto be construed as limiting the invention.

EXPERIMENTAL Differentiation of Adipose Tissue-Derived Stromal Cellsinto Chondrocytes Example 1 In Vitro Chondrogenesis Using Dexamethasone

Stromal cells are isolated from human subcutaneous adipose tissueaccording to methods described in “Methods and Composition of theDifferentiation of Human Preadipocytes into Adipocytes” Ser. No.09/240,029 filed Jan. 29, 1999. These cells are plated at a density of500 to 20,000 cells per cm². The present invention contemplates that thecreation of a precartilage condensation in vitro promotes chondrogenesisin mesenchymal progenitor cells derived from human adipose tissue. Thisis accomplished by methods including, but not limited to:

-   -   (1) The pellet culture system, which was developed for use with        isolated growth plate cells (Kato et al. (1988) PNAS        85:9552-9556; Ballock & Reddi, J. Cell Biol. (1994)        126(5):1311-1318) and has been used to maintain expression of        the cartilage phenotype of chondrocytes placed in culture        (Solursh (1991) J. Cell Biochem. 45:258-260).    -   (2) The alginate suspension method, where cells are maintained        in a calcium alginate suspension to prevent cell-cell contact        and maintain a characteristic rounded morphology promoting the        maintenance or acquisition of the chondrocyte phenotype.

Human adipose tissue-derived cells are isolated as described above. Forpellet cultures, aliquots of 200,000 cells were centrifuged at 500 g for10 minutes in sterile 15 ml conical polypropylene tubes in DMEM with 10%fetal bovine serum, 50 ng/ml ascorbate-2-phosphate, 100 nM dexamethasone(DEX) and then incubated at 37° C. in a 5% CO₂ incubator for up to 3weeks. For alginate cultures, cells were suspended at a density of 1million cells per ml in 1.2% calcium alginate and maintained in DMEMwith 10% fetal bovine serum, 50 ng/ml ascorbate-2-phosphate, 100 nMdexamethasone (DEX) and then incubated at 37° C. in a 5% CO₂ incubatorfor up to 3 weeks. After 2 or 4 weeks, the cells were isolated, fixedand analyzed for chondrocyte lineage markers by immunohistochemistrywith appropriate antibody reagents or by staining with toluidine blue todetect the presence of sulfated proteoglycans in the extracellularmatrix.

Results obtained with an antibody detecting a representative chondrocytemarker protein, collagen II, are shown in FIGS. 1-3. The cellsmaintained in pellet culture (FIG. 2) or calcium alginate (FIG. 3)stained positive by immunofluorescence for the intracellular presence ofthe collagen II protein. These results are to be contrasted withidentical analysis of adipose tissue-derived cells maintained for 3weeks in monolayer culture as shown in FIG. 1; here, no stainingwhatsoever is observed. Immunohistochemical results with an antibodyreagent detecting the chondrocyte marker protein, collagen VI, are shownin FIG. 4. Adipose tissue-derived stromal cells were maintained in 1.2%calcium alginate and maintained in DMEM with 10% fetal bovine serum, 50ng/ml ascorbate-2-phosphate, 100 nM dexamethasone (DEX) in the absenceor presence of transforming growth factor β (10 ng/ml) and thenincubated at 37° C. in a 5% CO₂ incubator for up to 2 weeks.Immunohistochemistry revealed a dense deposition of the collagen VIprotein surrounding those cells maintained in the presence, but not theabsence, of transforming growth factor β.

Polymerase chain reaction results detecting representative gene markersassociated with chondrogenesis are shown in FIG. 5. Adiposetissue-derived stromal cells were maintained in 1.2% calcium alginate(Alg) or in monolayer (Mono) cultures and maintained in DMEM with 10%fetal bovine serum, 50 ng/ml ascorbate-2-phosphate, 100 nM dexamethasone(DEX) in the absence (TGFβ−) or presence (TGFβ+) of transforming growthfactor β (10 ng/ml) for a period of 4 weeks. Total RNA was isolated fromthe individual cultures and used in polymerase chain reactions withprimers specific for collagens types I or VI, the proteoglycan link(Link) protein, aggrecan, or actin. The collagen markers and actin weredetected under all growth conditions. However, the link mRNAs were mostabundant under alginate suspension conditions and aggrecan was onlypresent under alginate conditions in the presence of TGFβ.

These results demonstrate that, through a combination of creating an invitro cell condensation and adding the appropriate permissive factors,we are able to produce the expression of chondrocyte markers consistentwith chondrogenesis in cells from subcutaneous adipose tissue.

Example 2 Preparation of Synthetic Cartilage Patch

Following proliferation, the chondrogenic cells still havingchondrogenic potential may be cultured in an anchorage-independentmanner, i.e., in a well having a cell contacting, cell adhesive surface,in order to stimulate the secretion of cartilage-specific extracellularmatrix components.

Heretofore, it has been observed that chondrogenic cells proliferativelyexpanded in an anchorage-dependent manner usually dedifferentiate andlose their ability to secrete cartilage-specific type II collagen andsulfated proteoglycan. (Mayne et al. (1984) Exp. Cell. Res. 151(1):171-82; Mayne et al. (1976) PNAS 73(5): 1674-8; Okayama et al. (1976)PNAS 73(9):3224-8; Pacifici et al. (1981) J. Biol Chem. 256(2): 1029-37;Pacifici et al. (1980) Cancer Res. 40(7): 2461-4; Pacifici et al. (1977)Cell 4: 891-9; von der Mark et al. (1977) Nature 267(5611):531-2; Westet al. (1979) Cell 17(3):491-501; Oegama et al. (1981) J. Biol. Chem.256(2):1015-22; Benya et al. (1982) Cell 30(1):215-24).

It has been discovered that undifferentiated chondrogenic cells, whenseeded into, and cultured in a well having a cell contacting surfacethat discourages adhesion of cells to the cell contacting surface, thecells redifferentiate and start to secrete cartilage-specific collagenand sulfated proteoglycans thereby to form a patch of syntheticcartilage in vitro (U.S. Pat. Nos. 5,902,741 and 5,723,331).

In addition, it has been found that culturing the cells in a pre-shapedwell enables one to manufacture synthetic cartilage patches ofpredetermined thickness and volume. It is appreciated, however, that thevolume of the resulting patch of cartilage is dependent not only uponthe volume of the well but also upon the number of chondrogenic cellsseeded into the well. Cartilage of optimal predetermined volume may beprepared by routine experimentation by altering either or both of theaforementioned parameters.

A. Preparation of Pre-Shaped Well

Several approaches are available for preparing pre-shaped wells withcell contacting, cell adhesive surfaces.

The cell contacting surface of the well may be coated with a moleculethat discourages adhesion of chondrogenic cells to the cell contactingsurface. Preferred coating reagents include silicon based reagents i.e.,dichlorodimethylsilane or polytetrafluoroethylene based reagents, i.e.,TEFLON®. Procedures for coating materials with silicon based reagents,specifically dichlorodimethylsilane, are well known in the art. See forexample, Sambrook et al. (1989) “Molecular Cloning A Laboratory Manual,”Cold Spring Harbor Laboratory Press, the disclosure of which isincorporated by reference herein. It is appreciated that otherbiocompatible reagents that prevent the attachment of cells to thesurface of the well may be useful in the practice of the instantinvention.

Alternatively, the well may be cast from a pliable or moldablebiocompatible material that does not permit attachment of cells per se.Preferred materials that prevent such cell attachment include, but arenot limited to, agarose, glass, untreated cell culture plastic andpolytetrafluoroethylene, i.e., TEFLON®. Untreated cell culture plastics,i.e., plastics that have not been treated with or made from materialsthat have an electrostatic charge are commercially available, and may bepurchased, for example, from Falcon Labware, Becton-Dickinson, LincolnPark, N.J. The aforementioned materials, however, are not meant to belimiting. It is appreciated that any other pliable or moldablebiocompatible material that inherently discourages the attachment ofchondrogenic cells may be useful in the practice of the instantinvention.

The size and shape of the well may be determined by the size and shapeof the articular cartilage defect to be repaired. For example, it iscontemplated that the well may have a cross-sectional surface area of 25cm². This is the average cross-sectional surface area of an adult, humanfemoral chondyle. Accordingly, it is anticipated that a single piece ofsynthetic cartilage may be prepared in accordance with the invention inorder to resurface the entire femoral chondyle. The depth of the well ispreferably greater than about 0.3 cm and preferably about 0.6 cm indepth. The thickness of natural articular cartilage in an adultarticulating joint is usually about 0.3 cm. Accordingly, the depth ofthe well should be large enough to permit a cartilage patch of about 0.3cm to form. However, the well should also be deep enough to containgrowth medium overlaying the cartilage patch.

It is contemplated also that a large piece of cartilage prepared inaccordance with the invention may be “trimmed” to a pre-selected sizeand shape by a surgeon performing surgical repair of the damagedcartilage. Trimming may be performed with the use of a sharp cuttingimplement, i.e., a scalpel, a pair of scissors or an arthroscopic devicefitted with a cutting edge, using procedures well known in the art.

The pre-shaped well preferably is cast in a block of agarose gel underaseptic conditions. Agarose is an economical, biocompatible, pliable andmoldable material that can be used to cast pre-shaped wells, quickly andeasily. As mentioned above, the dimensions of the well may be dependentupon the size of the resulting cartilage plug that is desired.

A pre-shaped well may be prepared by pouring a hot solution of molten LTagarose (BioRad, Richmond, Calif.) into a tissue culture dish containinga cylinder. The cylinder having dimensions that mirror the shape of thewell to be formed. The size and shape of the well may be chosen by theartisan and may be dependent upon the shape of the articular cartilagedefect to be repaired. Once the agarose has cooled and solidified aroundthe cylinder, the cylinder is carefully removed with forceps. Thesurface of the tissue culture dish that is exposed by the removal of thecylinder is covered with molten agarose. This seals the bottom of thewell and provides a cell adhesive surface at the base of the well. Whenthe newly added molten LT agarose cools and solidifies, the resultingpre-shaped well is suitable for culturing, and stimulating theredifferentiation of proliferated chondrogenic cells. It is appreciated,however, that alternative methods may be used to prepare a pre-shapedwell useful in the practice of the invention.

B. Growth of Cartilage Patch

Proliferated chondrogenic cells in suspension may be seeded into andcultured in the pre-shaped well. The cells may be diluted by theaddition of cell culture medium to a cell density of about 1×10⁵ to1×10⁹ chondrogenic cells per ml. A preferred cell culture mediumcomprises DMEM supplemented with 10% fetal bovine serum.

Within about four hours of seeding the chondrogenic cells into the well,the cells may coalesce to form a cohesive plug of cells. After about4-10 days, the cells will start to secrete cartilage-specific sulfatedproteoglycans and type II collagen. After prolonged periods of time inculture the collagen expressed by the chondrogenic cells in the wellwill be predominantly type II collagen. It is contemplated, however,that the cohesive plug of cells formed within four hours may be removedfrom the well and surgically implanted into the cartilage defect. It isanticipated that the undifferentiated chondrogenic cells subsequentlymay redifferentiate in situ thereby to form synthetic cartilage withinthe joint.

It is contemplated that chondrocytic differentiation or stimulatoryfactors may be added to the chondrogenic cells in the pre-shaped well toenhance or stimulate the production of articular cartilage specificproteoglycans and/or collagen (Luyten & Reddi (1992) in “BiologicalRegulation of the Chondrocytes,” CRC Press, Boca Raton, Ann Arbor,London, and Tokyo, pp. 227-236). Preferred growth factors include, butare not limited to transforming growth factor-β (TGF-β), insulin-likegrowth factor (IGF), platelet derived growth factor (PDGF), epidermalgrowth factor (EGF), acidic fibroblast growth factor (aFBF), basicfibroblast growth factor (bFBF), hepatocytic growth factor (HGF),keratinocyte growth factor (KGF), the bone morphogenic factors (BMPs)i.e., BMP-1, BMP-2, BMP-3, BMP-4, BMP-5 and BMP-6 and the osteogenicproteins (OPs), i.e., OP-1, OP-2 and OP-3. Preferred concentrations ofTGF-β, IGF, PDGF, EGF, aFBF, bFBF, HGF, and KGF, range from about 1 to100 ng/ml. Preferred concentrations of the BMP's and OP's range fromabout 1 to about 500 ng/ml.

However, these particular growth factors are not limiting. Anypolypeptide growth factor capable of stimulating or inducing theproduction of cartilage specific proteoglycans and collagen may beuseful in the practice of the instant invention.

In addition, it is contemplated that ascorbate may be added to thechondrogenic cells in the pre-shaped well to enhance or stimulate theproduction of cartilage specific proteoglycans and collagen. Preferredconcentrations of ascorbate range from about 1 to about 1000 μg/ml.

Example 3 Surgical Repair of Articular Cartilage Defect

Cartilage defects in mammals are readily identifiable visually duringarthroscopic examination or during open surgery of the joint. Cartilagedefects may also be identified inferentially by using computer aidedtomography (CAT scanning), X-ray examination, magnetic resonance imaging(MRI), analysis of synovial fluid or serum markers or by any otherprocedures known in the art. Treatment of the defects can be effectedduring an arthroscopic or open surgical procedure using the methods andcompositions disclosed herein.

Accordingly, once the defect has been identified, the defect may betreated by the following steps of (1) surgically implanting at thepredetermined site, a piece of synthetic articular cartilage prepared bythe methodologies described herein, and (2) permitting the syntheticarticular cartilage to integrate into predetermined site.

The synthetic cartilage patch optimally has a size and shape such thatwhen the patch is implanted into the defect, the edges of the implantedtissue contact directly the edges of the defect. In addition, thesynthetic cartilage patch may be fixed in place during the surgicalprocedure. This can be effected by surgically fixing the patch into thedefect with biodegradable sutures, i.e. (Ethicon, Johnson & Johnson),and/or by applying a bioadhesive to the region interfacing the patch andthe defect. Preferred bioadhesives include, but are not limited to:fibrin-thrombin glues similar to those disclosed in Fr. Pat. No. 2 448900; Fr. Pat. No. 2 448 901 and EP.S.N. 88401961.3 and syntheticbioadhesives similar to those disclosed in U.S. Pat. No. 5,197,973. Itis contemplated, however, that alternative types of sutures andbiocompatible glues may be useful in the practice of the invention.

In some instances, damaged articular cartilage may be surgically excisedprior to the implantation of the patch of synthetic cartilage.Additionally, the adhesion of the synthetic cartilage patch to thearticular cartilage defect may be enhanced by treating the defect withtransglutaminase (Ichinose et al. (1990) J. Biol. Chem. 265(3):13411-13414; Najjar et al. (1984) in “Transglutaminases,” Boston,Martinuse-Nijhofi). Initially, the cartilage defect is dried, forexample, by using cottonoid, and filled with a solution oftransglutaminase. The solution is subsequently removed, for example, byaspiration, leaving a film containing transglutaminase upon thecartilage. The synthetic cartilage patch is implanted subsequently intothe defect by the methods described above.

In addition the synthetic cartilage may be useful in the repair of humanarticular cartilage defects. Accordingly, chondrogenic cells may bedifferentiated from human adipose tissue-derived stromal cells, i.e.,human subcutaneous adipose tissue.

Surgical procedures for effecting the repair of articular cartilagedefects are well known in the art. See, for example: Luyten & Reddi(1992) in “Biological Regulation of the Chondrocytes,” CRC Press, BocaRaton, Ann Arbor, London, & Tokyo, pp. 227-236, the disclosure of whichis incorporated by reference herein.

The above demonstrates a culture system in which human adiposetissue-derived stromal cells differentiate into hypertrophicchondrocytes. Since all components are defined, the system can be usedfor studies of the effects of growth factors, etc. on the progression ofchondrogenesis. In vitro systems have been used by us and others to showthat these cell populations have osteogenic and adipocytic potential. Wedemonstrate here that this population has chondrogenic potential. Thishas clinical applicability for cartilage repair.

The invention also provides a process for inducing chondrogenesis inhuman adipose tissue-derived stromal cells by contacting such cells witha chondroinductive agent in vitro where the stromal cells are associatedin a three dimensional format.

The invention also provides a process for using in vitro differentiatedchondrocytes from adipose-derived stromal cells in the repair ofcartilage tissue in mammals, including humans.

In the above methods, the stromal cells are preferably isolated, cultureexpanded human adipose tissue-derived stromal cells in a chemicallydefined environment and are condensed into close proximity, such as inthe form of a three-dimensional cell mass, e.g., packed cells or acentrifugal cell pellet. Further, the contacting preferably comprisesculturing a pellet of human adipose tissue-derived stromal cells in achemically defined medium which comprises DMEM with 10% serum, 50 ng/mlascorbate-2-phosphate, 10⁻⁷ M dexamethasone. The differentiated cellsare then introduced into the surgery site to repair cartilage. Since allcomponents of the system are defined, the system can be used as aproduct for cartilage repair in mammals, including man and horses.

What is claimed is:
 1. A method for differentiating adipose tissuederived stromal cells into chondrocytic cells, comprising: a) pelletingsaid stromal cells by centrifuging between 50,000 to 5 million cells at500×g for 2 to 20 minutes in sterile tubes containing a medium such asDulbecco's Modified Eagle's Medium (DMEM) or alpha modified MinimalEssential Medium (αMEM) or Roswell Park Memorial Institute media 1640(RPMI Media 1640); b) plating isolated stromal cells at a density of 500to 20,000 cells/cm² in a differentiating medium; c) supplementing saidmedium with: (i) a chondroinductive agent capable of activating anycellular signal transduction pathway leading to the mature chondrocytephenotype; (ii) an antibiotic; (iii) a nutrient supplemented with 1 to20% fetal bovine serum or 1 to 20% horse serum or any other biologicalor synthetic equivalent combination of proteins; (iv) ascorbate orrelated vitamin C analog; (v) a glucocorticoid or other chemical agentcapable of activating the cellular glucocorticoid receptor; and d)incubating said cells at about 31° C. to 37° C. for about 3-4 weeks inwith 5% CO₂ and between 1% and 20% oxygen.
 2. A method fordifferentiating adipose tissue derived stromal cells into chondrocyticcells, comprising: a) suspending stromal cells at a concentration of 0.5to 10 million cells per ml in calcium alginate or any otherbiocompatible lattice or matrix of supporting chondrogenesis in athree-dimensional configuration; b) transferring cells to 35 mm culturedishes and plating cells at a density of 500 to 20,000 cells/cm² in adifferentiating medium comprising a chemically defined culture mediumhaving or supplemented with: (i) a chondroinductive agent capable ofactivating any cellular transduction pathway leading to the maturechondrocyte phenotype; (ii) an antibiotic; (iii) a nutrient supplementedwith 1 to 20% fetal bovine serum or 1 to 20% horse serum or any otherbiological or synthetic equivalent combination of proteins; (iv)ascorbate or related vitamin C analog; (v) a glucocorticoid or otherchemical agent capable of activating the cellular glucocorticoidreceptor; and (c) incubating said cells at about 31 to 37° C. for about3-4 weeks in an incubator with 5% CO₂ and between 1% and 20% oxygen.